Loss of Sfrp1 increases Wnt signaling and cellular proliferation in response to DIO the murine mammary gland. (A) Total RNA was harvested from 5th inguinal mammary glands and employed for real-time PCR analysis of Myc gene expression (n = 6/genotype). The results shown represent experiments performed in duplicate and are normalized to the amplification of β-Actin mRNA. Bars represent mean ± SEM of the difference in fold change compared with control ND fed mice. (B) Upper panel, Mammary gland lysates were analyzed for non-phospho (active) β-catenin and β-actin protein expression by western blot. Lower panel, Band density was quantified and bars represent mean ± SEM of % control ND fed mice. (C) Left panel, 3rd & 4th inguinal mammary gland sections were subjected to immunohistochemical analysis, stained for BrdU (brown chromogen), and representative images were captured at 400X are displayed for mice in each treatment group (scale bar 50 μm). Right panel, BrDU-stained cells were counted out for each mammary gland (n = 6/genotype) and bars represent mean ± SEM % BrdU-positive cells. (*p < 0.05, significantly different from control mice fed a ND using Bonferroni’s t test after a two-way ANOVA).